Many methods have been used for removing the red blood cells from whole blood so that the leukocytes can be studied by flow techniques. Physical separation by sedimentation or centrifugation or density gradients, aggregation of red blood cells and other physical techniques are useful for research purposes, but are too slow and difficult for automated clinical analysis of leukocytes. Quaternary ammonium salt detergents are very efficient lytic agents, but have been found to be too damaging to the leukocytes, resulting at best, in only three clusters of leukocytes, by DC volume analysis, representing the lymphocytes, monocytes and granulocytes.
Kim, U.S. Pat. No. 4,099,917, 1978, describes a method of sensitizing red blood cells with a non-ionic detergent, adding a formaldehyde fixative, and incubating the blood at 58.degree. C., to lyse the red blood cells selectively, leaving leukocytes and platelets intact for light scatter measurements. This process is rather slow, about three minutes, and may be sufficient to make the red blood cells transparent toward optical measurements, but not towards electronic measurements, which require more thorough stromatolyzing of the red blood cells. The same is true of other lytic procedures, such as hypotonic lysis, ammonium chloride lysis, and ethylene or propylene glycol treatment which render the red blood cells transparent towards optical, i.e. light scatter, fluorescence measurements, but not towards electronic, i.e. DC volume and R.F. volume parameters.
The natural product known as saponin has long been used as a red blood cell lytic agent. Saponin is chemically defined as a class of glycosides of various mono- or polysaccharides, with steroid or triterpene alcohols. Quillaja saponin is isolated as a natural product from quillaja tree bark. This saponin has detergent-like properties and hemolyzes red blood cells when used at very low concentrations compared to synthetic ionic and nonionic hemolytic agents. However, the chemical treatment, structure and purity of commercial quillaja saponin is not generally specified or tested, and the material does vary from lot to lot.
The activity of saponin is more selective towards red blood cells than are the quaternary ammonium salt detergents. Unfortunately, by employing lysing procedures known heretofore, it has not been possible to obtain leukocytes free from red blood cells, without doing some concomitant damage to the leukocytes.
Several reports describe the use of saponin with a second reagent which retards the leukocyte damage. Hughes-Jones, J. Clin. Path., Vol. 27, page 623 (1974), reported a treatment of diluted whole blood with a saponin solution, followed in three minutes by treatment with serum which quenches the saponin activity. A direct current volume analysis was done on the leukocytes. Ornstein, Blood Cells, Vol. 25, page 57 (1967), used a solution of saponin, formaldehyde and other components, for twenty seconds followed by enzyme staining for detecting various leukocyte types. Humphries et al, Ser. Haemat. Vol. V-2, page 142 (1972), treat diluted whole blood with saponin, followed in thirty seconds with dilution in cold phosphate buffered saline to quench the lytic action. Other reports are Ladinsky, Cancer Res., Vol. 27, page 1689 (1967), and Van Dilla, Proc. Soc. Exp. Biol. (NY), Vol. 125, page 367 (1967).
Commercial equipment employing the teachings of U.S. Pat. Nos. 2,656,508; and 3,259,842 are known under the trademark COULTER COUNTER.RTM., and the principle of their operation is commonly known as the Coulter principle.
According to the Coulter principle, first patented in U.S. Pat. No. 2,656,508, 1953, where a particle of microscopic size is passed through an electrical field of small dimensions of an order approaching those of a particle, there will be a momentary change in the electric impedance. If the electrical field is excited by a direct (DC) or low frequency current, the electrical change is closely proportional to the volume of the particle. In commercial apparatus, the changes are detected by some suitable means and used to operate counters and analyzers. The analyzers associated with such apparatus classify and size particles into populations based upon particle volume and record the data obtained.
The invention was materially expanded in U.S. Pat. No. 3,502,974, Coulter et al, 1970, using radio frequency (RF) current in addition to DC current field excitation to provide not only DC volume information concerning the particle studied, but also information due to the composition and nature of the material constituting the particle. This patent discloses apparatus capable of distinguishing between particles of identical size, but of different material. By generating the particle sensing field by means of both a low frequency or direct current (DC) and radio frequency (RF) current excitation, two or more interrelated output signals can be derived from the passage of a single particle through the electrical field. This is due to the fact that, although the subject particles are nearly always insulators with respect to low frequency or direct current fields, they are capable of carrying or impeding radio frequency current differently from the surrounding electrolyte. This may be due to differences in the dielectric constant in the case of homogeneous particles, or to the sac-like structure in the case of blood cells which have, enclosed in an extremely thin membrane, contents having conductivities different from the electrolyte. Thus, while all the DC current goes around a blood cell, some of the RF current will go through it. The ease with which the RF current will go through a particle is a measure of what is termed its "electrical transparency", or simply "transparency", in analogy with light transmission; whereas, a particle's ability to impede RF current is termed its "opacity". In later publications, "opacity" is defined as the RF impedance divided by the DC impedance.
The relative electrical opacity of a particle becomes an identifying feature of the particle contents, and hence, its particle type for classification purposes. To the extent that different types of particles each possess a different opacity, the difference between them is detectable. However, significantly different particles can possess substantially the same opacity and such particles cannot be classified effectively in this manner. In U.S. Pat. No. 3,836,849, 1974, Coulter et al taught that it is possible to change selectively the opacity of particle types by treatment of the particles, so that detectable differences result.
Although red blood cells and white blood cells nominally have different sizes, their size ranges tend to overlap, or at least under certain conditions of health could overlap. Moreover the opacities of these two types of blood cells may also overlap.
U.S. Pat. No. 3,741,875, Ansley et al, June, 1973, describes a process for obtaining a differential white blood cell count. A cytological fixing agent, which is a monoaldehyde such as formaldehyde, is added to a blood sample. A hemolyzing agent then is added after the fixation step to cause the red blood cells to release their hemoglobin content into solution. Addition of a specific cytochemical substrate, chromogenic precipitating coupling reagent, and pH buffer causes deposition of an insoluble dye in a specific type of cell containing an immobilized enzyme. The solution containing the dyed blood cells then is passed through a photometric counter. Using different specific substrates for different enzymes contained in specific kinds of cells, absolute and relative counts of the different kinds of cells are obtained. The cytological fixing solution utilized only a monoaldehyde. Dialdehydes are stated to be unsuitable, since they cross-link and produce extracellular precipitates.
Starting with whole blood, it is necessary to hemolyze the red blood cells, since there is danger that coincident passage of two or more red cells through a photometric counting station could be mistaken for dyed white blood cells or abnormal cells. A preferred way to solve the problem is to hemolyze the red blood cells by addition of a reagent to the suspension of cells to cause the red blood cells to rupture and release their hemoglobin content into the solution.
Ledis et al, U.S. Pat. Nos. 4,286,963, 1981, teaches a method for two-volume analysis of leukocytes using a COULTER COUNTER analyzer which employs only DC field excitation instrumentation and quaternary ammonium salts as lysing agents.
Ledis et al, U.S. Pat. No. 4,485,175, 1984, to Coulter Electronics, Inc. concerns a method and reagent system for three-volume differential determination of lymphocyte, monocyte, and granulocyte populations of leukocytes, using quaternary ammonium salts as lysing agents and the COULTER COUNTER Model S Plus automated blood counter, which instrument employs only direct current field excitation.
Previous methods of flow analysis of leukocytes using DC volume, or light scatter at various angles have shown only three clusters of leukocytes, corresponding to lymphocytes, monocytes, and granulocytes including neutrophils and eosinophils. The eosinophils have been observed as a distinct cluster by using special fluorescence techniques.
Other dye compositions for differential analysis of white blood cells include a hypotonic aqueous solution of a metachromatic fluorochrome dye such as acridine orange, Adams, U.S. Pat. No. 3,883,247. The white cell analysis is made by suspending a sample of fresh blood in the dye solution, subjecting the suspension, before dye uptake equilibrium is reached, to radiation from a light source, e.g. radiation from a blue laser, having a wave length within the range of absorption of the dye, and distinguishing the white cells from other blood particles by detecting fluorescences, e.g. green vs. red fluorescences.
Fluorescent dyes suitable for specifically dyeing eosinophil granules are the anilino or toluidino naphthalene sulfonic acids and their alkyl, alkoxy or halogen substituted derivatives.
The development of instrumentation and fluorochromes for automated multiparameter analysis of cells is further described by R. C. Leif et al. in Clinical Chemistry, Vol. 23, pp 1492-98 (1977).
Eosinophils have been observed also by enzyme staining such as by the Technicon Hemalog D and H6000 instruments, (Ansley and Ornstein, Adv. Automated Anal., Vol. 1 437 (1971), Kaplow, Macrophages and Lymphocytes, Part A, 221 Plenum (1980).
The detection of populations of particular leukocytes, and the concurrent relationship of these populations to one another in a human blood sample is important in medical research and for the diagnosis of certain human diseases. Such data are useful as a screening tool for calling attention to abnormal leukocyte ratios. Abnormal situations identified by this method give information of diagnostic significance and alert the technologist to the need for further study.